Bioorthogonal cleavage chemistry (BCC) has been extensively applied to fluorescence-based imaging in cancer diagnostics. Its potential in chemiluminescence imaging is to be explored. In this study, a smart ruthenium (Ru)-catalyzed bioorthogonal activation chemiluminescence (BAC) probe is developed by integrating BCC with a phenoxy-adamantyl-1,2-dioxetane (PAD) for real-time, in vivo imaging of thiol-containing metabolites, particularly hydrogen sulfide (H 2S), associated with thiol dysregulation in the tumor microenvironment. The BAC probe overcomes many limitations existed in other chemiluminescence probes via a highly selective “Ru-locked” mechanism to achieve light-independent, thiol-triggered activation in complex tumor microenvironment. This mechanism enables rapid activation (1 min), high sensitivity (LOD = 0.243 μM), and stable luminescence with a half-life of 18.5 h, as determined in vitro, across a broad emission range (400-800 nm). The probe also demonstrates enhanced selectivity for thiol-containing metabolites, particularly H 2S, and exhibits low toxicity both in vitro and in vivo. In a breast cancer mouse model, the probe successfully visualizes endogenous H 2S with high spatial precision, supporting its utility in tumor localization and image-guided surgery. In addition, the PAD scaffolds are developed via an efficient synthetic route, significantly lowering production costs (300- to 400-fold) and increasing yields from 40% to 95%. Furthermore, our BAC probe holds a broad potential for non-invasive diagnosis and real-time monitoring of thiol dysregulation and pathophysiological processes.
